1. Field of the Invention
This invention relates to a measuring apparatus such as a surface plasmon resonance sensor for analyzing a material in a sample on the basis of generation of surface plasmon, and to a sensor unit for the measuring apparatus.
2. Description of the Related Art
In metal, free electrons vibrate in a group to generate compression waves called plasma waves. The compression waves generated in a metal surface are quantized into surface plasmon.
There have been proposed various surface plasmon resonance sensors for quantitatively analyzing a material in a sample utilizing a phenomenon that such surface plasmon is excited by light waves. Among those, one employing a system called “Kretschmann configuration” is best known. See, for instance, Japanese Unexamined Patent Publication No. 6(1994)-167443.
The surface plasmon resonance sensor using the Kretschmann configuration basically comprises a dielectric block shaped, for instance, like a prism, a metal film which is formed on one face of the dielectric block and is brought into contact with a sample, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at the interface of the dielectric block and the metal film and various angles of incidence of the light beam to the interface of the dielectric block and the metal film including an angle of incidence at which attenuation in total internal reflection is generated due to surface plasmon resonance (the attenuation angle) can be obtained, and an information obtaining means which detects the intensity of the light beam reflected in total internal reflection at the interface and obtains information on the attenuation angle and the change thereof.
In order to obtain various angles of incidence of the light beam to the interface, a relatively thin incident light beam may be caused to impinge upon the interface changing the angle of incidence or a relatively thick incident light beam may be caused to impinge upon the interface in the form of convergent light or divergent light so that the incident light beam includes components impinging upon the interface at various angles. In the former case, the light beam which is reflected from the interface at an angle which varies as the angle of incidence changes may be detected by a photodetector which is moved in synchronization with the change of the angle of incidence or by an area sensor extending in the direction in which reflected light beam is moved as the angle of incidence changes. In the latter case, an area sensor which extends in directions so that all the components of light reflected from the interface at various angles can be detected by the area sensor may be used.
In such a surface plasmon resonance sensor, when a light beam impinges upon the metal film at a particular angle of incidence θsp not smaller than the angle of total internal reflection, evanescent waves having an electric field distribution in the sample in contact with the metal film are generated and surface plasmon is excited in the interface between the metal film and the sample. When the wave vector of the evanescent light is equal to the wave number of the surface plasmon and wave number matching is established, the evanescent waves and the surface plasmon resonate and light energy is transferred to the surface plasmon, whereby the intensity of light reflected in total internal reflection at the interface of the dielectric block and the metal film sharply drops. The sharp intensity drop is generally detected as a dark line by the photodetector.
The aforesaid resonance occurs only when the incident light beam is p-polarized. Accordingly, it is necessary to set the surface plasmon sensor so that the light beam impinges upon the interface in the form of p-polarized light or p-polarized components are only detected.
When the wave number of the surface plasmon can be known from the angle of incidence θsp at which the phenomenon of attenuation in total internal reflection (ATR) takes place, the dielectric constant of the sample can be obtained. That is,
            K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in a vacuum, and ∈m and ∈s respectively represent the dielectric constants of the metal and the sample.
When the dielectric constant ∈s of the sample is known, the concentration of a specific material in the sample can be determined on the basis of a predetermined calibration curve or the like. Accordingly, the specific material in the sample can be quantitatively analyzed by detecting the angle of incidence θsp at which the intensity of light reflected in total internal reflection from the interface of the prism and the metal film sharply drops (this angle θsp is generally referred to as “the attenuation angle θsp”).
As a similar apparatus utilizing the phenomenon of attenuation in total internal reflection (ATR), there has been known a leaky mode sensor described in, for instance, “Surface Refracto-Sensor using Evanescent Waves: Principles and Instrumentations” by Takayuki Okamoto (Spectrum Researches, Vol. 47, No. 1 (1998), pp21 to 23 & pp26 and 27). The leaky mode sensor basically comprises a dielectric block shaped, for instance, like a prism, a clad layer which is formed on one f ace of the dielectric block, an optical waveguide layer which is formed on the clad layer and is brought into contact with a sample, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at the interface of the dielectric block and the clad layer and attenuation in total internal reflection is generated due to optical waveguide mode excitation can be obtained, and an information obtaining means which detects the intensity of the light beam reflected in total internal reflection at the interface and obtains information on the state of waveguide mode excitation (the attenuation angle and the change thereof.
In the leaky mode sensor with this arrangement, when the light beam is caused to impinge upon the clad layer through the dielectric block at an angle not smaller than an angle of total internal reflection, evanescent waves are generated in the optical waveguide layer and an evanescent wave having a particular wave number comes to propagate through the optical waveguide layer in a waveguide mode. When the waveguide mode is thus excited, almost all the incident light which generates the evanescent wave having a particular wave number is taken in the optical waveguide layer and accordingly, the intensity of light reflected in total internal reflection at the interface of the dielectric block and the clad layer sharply drops. That is, attenuation in total internal reflection occurs. Since the wave number of light to be propagated through the optical waveguide layer depends upon the refractive index of the sample on the optical waveguide layer, the refractive index and/or the properties of the sample related to the refractive index can be detected on the basis of the attenuation angle θsp at which the attenuation in total internal reflection occurs.
Such a measuring apparatus is employed, as a biosensor, to analyze a sample, that is, a sensing medium (e.g. antibody), which combines with a particular material (e.g., antigen), is disposed on the thin film (the metal film in the case of a surface plasmon resonance sensor, and optical waveguide layer in the case of a leaky mode sensor) and whether the sample includes a material combined with the sensing medium or the state of combination of the sample with the sensing medium is detected. As a method of analyzing a sample in this way, there has been proposed a method in which, in order to eliminate the influence of the solvent in the sample liquid on the refractive index of the sample liquid, refractive index information on buffer (the same as the solvent) free from the analyte (material to be analyzed) is first obtained and then the sample liquid is dispensed to the buffer to measure the refractive index information of the mixture after the reaction, whereby only the reaction of the analyte is precisely extracted.
As the surface plasmon resonance sensor, there have been known various types of sensors, as well as those in which the attenuation angle is detected, such as those in which light beams of different wavelengths are caused to impinge upon the interface and the degree of attenuation in total internal reflection is detected by the wavelength, or in which a light beam is caused to impinge upon the interface and a part of the light beam is split before the light beam impinges upon the interface and caused to interfere with the other part of the light beam reflected at the interface, thereby measuring the state of interference. Any one of the sensors is a sensor which indirectly obtains information on the refractive index of the analyte on the thin film and the change thereof and analyzes the analyte.
In order to increase efficiency of handling, for instance, in changing the sample in the measuring apparatus, there has been proposed in U.S. Patent Laid-Open No. 20010040680 a sensor well unit comprising a dielectric block, a thin film disposed on the upper surface of the dielectric block and a sample holding portion for holding the sample on the thin film, which are formed integrally with each other. The sensor unit is formed by providing a unit body in the form of a dielectric block with a sample well (sample holding portion) open in the upper surface, and by providing a film layer on the inner bottom surface of the sample well, and the part of the well body below the sample well functions as the known dielectric block which performs the duty of the light beam input-out system. In order to perform measurement on a number of samples at high speed, and to further increase efficiency of handling, there has been proposed a sensor unit formed by providing a unit body in the form of a bar-like or plate-like dielectric block with a plurality of one-dimensionally or two-dimensionally arranged sample wells open in the upper surface. A plurality of light beams are caused to impinge upon the plurality of sample wells in parallel and the reflected light reflected at the interface of each of the sample wells is separately detected.
It is sometimes necessary to perform measurement a plurality of times on a sample at intervals and to detect the change of the state. In such a case, in order to perform such measurement on a plurality of samples at high efficiency, there sometimes employed batch processing in which a first sensor unit is once demounted from the measuring portion (sensor holding portion) of a measuring apparatus after a first measurement on the sample placed in its sample well, another or a second sensor unit is mounted on the measuring portion of the measuring apparatus, and then the first sensor unit is mounted again on the measuring portion of the measuring apparatus after measurement on the samples placed in the sample wells of the second senor unit. Conventionally, there has been a problem that the position of the interface changes each time the same sensor unit is mounted on the measuring portion, which can result in a measuring error.
As a method of dealing with vertical displacement of the interface, there has been proposed a method in which vertical displacement of the outer bottom surface of the sensor unit is measured with the outer bottom surface of the sensor unit taken as a reference plane, and the vertical position of the sensor unit is adjusted on the basis of the vertical displacement of the outer bottom surface of the sensor unit.
However, these inventor's investigation has revealed that even if the vertical position of the sensor unit is adjusted on the basis of the vertical displacement of the outer bottom surface of the sensor unit, there remains a measuring error (an error produced when the state of light reflected in total internal reflection is measured) due to vertical displacement of the interface.
That is, the difference in the vertical direction between the position of the outer bottom surface of the sensor unit and the position of the interface (which is an actual surface of measurement) causes an error in measurement of the vertical position of the interface due to thermal expansion of the sensor unit in response to change in temperature. The distance between the outer bottom surface of the sensor unit and the inner bottom surface of the well in the sensor unit actually used in the measuring apparatus (distance b in FIG. 1B) is 6.7 mm. In this case, the vertical position of the inner bottom surface of the well is deviated from the position determined on the basis of the position of the outer bottom surface of the sensor unit by 0.6 μm/° C., which corresponds to shift in reflection angle of the reflected light of 0.0004° (4 ORU).